Classical planar thin layer chromatography (TLC) was introduced over 50 years ago. Izmailov et al., Farmatsiya 3, 1 (1938). Planar TLC is the method of choice for the separation and isolation of many classes of lipid compounds, especially those found in mammalian cell membranes: glycosphingolipids, phospholipids, neutral glycerol derivatives containing acyl chains such as triglycerides and diglycerides, and cholesteryl esters.
Recent advances in TLC, especially with regard to the reduction in, and uniformity of, silica particle size and the uniformity of plate coating, have led to the production of high performance TLC plates which significantly enhance separations of analytes. But the current TLC methods are problematic both in quantification and in analyzing significant mass changes in multiple samples of smaller cellular lipids, such as phosphatidic acid (PA) and diradylglycerols (DG). Conventional methods simply lack high-throughput capability.
Although historically important by virtue of their role in membrane formation, lipids have become the focus of interest more recently for a variety of physiologically important reasons. For example, recent interest in PA and DG has been piqued by the discovery that they play important roles in cellular signaling. See, for example, English et al., Chem. Physics of Lipids 80, 117-132 (1996); Mathias et al., Advances in Lipid Research 25, 65-89 (1993); and Hannun et al., loc. cit. 43-64 (1993). PA species, for example, have been implicated in mitogenesis, cellular transformation, and exocytic processes. See English, Cell Signal 8, 341-347 (1996), and Boarder, Trends in Pharmacological Sciences, 15, 57-61 (1994). PA species also have been implicated in inflammatory signaling by Interleukin-1 (IL-1). Bi et al., Current Biology 7, 301-307 (1997). In addition, many different enzymes which regulate PA cellular concentration, are strongly associated with signaling by cell activators, including Lipid A and the cytokines IL-1 and IL-2. See Mollinedo et al., J. Immunol. 153, 2457-2462 (1994); Rice et al., Proc. Nat'l Acad. Sci USA 91, 3857-3861 (1994); and West et al., DNA and Cell Biology 16, 691-701 (1997). Among such enzymes are lyso-PA acyl CoA: acyltransferase (LPAAT), phosphatidate phosphohydrolase (PAP), diacylglycerol kinase (DG kinase), and phosphatidylcholine-directed phospholipase D (PC-PLD). The measurement of the substrates and products of these enzymes directly correlate to a physiological state.
DG species have been implicated in cell activation through protein kinase C (PKC) signaling, or otherwise regulating calcium-sensitive PKC isotypes, respectively. Musial et al., J. Biol. Chem. 270, 21632-21638 (1995); Baldi et al., J. Applied Physiol. 217, 356-365 (1994); Kester et al., J. Clin. Invest 83, 718-723 (1989); and Mandal et al., J. Biol. Chem 272, 20306-20311 (1997). DG participation in a variety of separable cellular synthetic pathways, like PA, has been implicated in different inflammatory processes. Mandal et al., J. Biol. Chem 272, 20306-20311 (1997), and Bursten et al., loc. cit. 266, 20732-20743 (1991).
It has become of increasing interest, therefore, to measure changes in mass in these lipid species and to isolate them for resolution and identification of component species and sub-species, such as by gas chromatographic analysis of acyl chain composition and/or mass spectrometry. Bursten et al., J. Biol. Chem. 266, 20732-20743 (1991); Rice et al., Proc. Nat'l Acad. Sci 91, 3857-3861 (1994); and Lavie et al., J. Biol. Chem. 271, 19530-19536 (1996).
Previous separation methods have not been adequate, however, to demonstrate an origin and/or acyl composition of these critical lipid species. These failings include: destructive detection methods which result in an inability to perform serial analyses; insufficient concentrations of lipids because of poor detection limits; and labeling methods which are insensitive and not specific enough, which leads to ambiguous, if not erroneous results.
Two-dimensional TLC systems are sometimes satisfactory for the separation of complex mixtures of lipids, but none allows for examination of multiple samples on the same plate. The use of one-step (single resolution) TLC systems permits the analysis of multiple samples, but lacks the ability to separate complex mixtures. TLC analysis of phospholipids such as PA also has heretofore lacked the ability to separate congeners of PA such as lyso(bis) PA, cardiolipin, (bis)PA, and/or phosphatidylglycerol, or has been frustrated by the small amounts of PA present or generated in different cell types. See Bursten et al., Cell Physiol. 35, C1093-C1104 (1994); Wen et al., Amer. J. Physiol 269 (Cell Physiol 38) C435-C442 (1995); Flores et al., J. Biol. Chem. 271, 10334-10340 (1996); Eardley et al., Science 251, 78-81 (1991); Musial et al., J. Biol. Chem. 270, 21632-21638 (1995); Booz Molecular and Cellular Biochemistry 141, 135-143 (1994); Huterer et al., J. Lipid Research 20, 966-973 (1979); Pannell et al., Biochimica et Biophysica Acta 1125, 330-334 (1992); and van Blitterswijk, EMBO J. 12, 2655-2662 (1993).
Previous quantification techniques typically have relied upon less sensitive TLC techniques. Some of these poor techniques, in turn, have depended upon radioactive labeling of cells using either selected acyl chains, such as .sup.14 C-myristate, or alkyl lyso-phospholipids, for example, .sup.14 C-alkyl-lyso-PC. Aside from the obvious radioactivity hazard and an inability to use such samples in mass determinations, such methods also are problematic. Labeling of cells with myristate, for example, works on the assumption of selective labeling of phosphatidylcholine, which may contain the bulk of myristate but may not be the source of PA, rendering such analysis irrelevant. Bursten et al., Cell Physiol. 35, C1093-C1104 (1994), and Eardley et al., Science 251, 78-81 (1991). In addition, selective labeling of PC with alkyl-lyso-PC may identify activation of PC-PLD, but it cannot identify simultaneous activation of the other PA-generating enzymes, such as DG kinase or LPAAT (9, 13, 20, 24, 30, 31). Bursten et al., J. Biol. Chem. 266, 20732-20743 (1991); Rice et al., Proc. Nat'l Acad. Sci USA 91, 3857-3861 (1994); Lavie et al., J. Biol. Chem. 271, 19530-19536 (1996); Bursten et al., Circulatory Shock 44, 14-29 (1994); Flores et al., J. Biol. Chem. 271, 10334-10340 (1996); and Eardley et al., Science 251, 78-81 (1991).
Many methods also rely on HPLC separation of cellular phospholipids. HPLC separation of phospholipids is confounded, however, by the tendency of PA to separate widely on silica columns by acyl and/or alkyl/alkenyl composition, but then co-migrate with similarly composed PA congeners such as cardiolipin. For example, dilinoleoyl PA is known to comigrate with cardiolipin, due to significant linoleoyl content of the latter. This necessitates serial gradient HPLC separations resulting in significant loss of mass, or requires very large, sometimes prohibitive, sample sizes to demonstrate significant lipid changes. Wen et al., Amer. J. Physiol 269 (Cell Physiol 38), C435-C442 (1995). These methods are expensive, inefficient, and cannot be applied to dose-responses, time courses, or other comparisons which must generate multiple data points for analyses in parallel.
A need exists, therefore, for a method facilitating the rapid, sensitive, and quantitative simultaneous resolution and detection of multiple biologically relevant molecules under a variety of conditions on the same plate, while maintaining lipid integrity for further analysis. This need encompasses improvement of methods for quantification of cellular lipids, including rare species of biologically-active cellular lipids, such as PA and DG. These methods should render lipids amenable to further analysis of hydrolyzed acyl chains by methods such as gas chromatography, which requires high quality separation. Importantly, these methods should be susceptible to high-throughput.